The tumor suppressor ARF is mutated or deregulated (by hypermethylation) in a variety of cancers. ARF is critical in the context of oncogenic activation and stress-induced senescence. It is believed that, following oncogenic activation, ARF induces senescence or apoptosis to protect cells from tumorigenic consequences. How does ARF dominate over the oncogenes? Previous studies suggested that the function of ARF is mediated through p53. However, recent genetic analyses provided evidence for additional p53-independent activities of ARF. Studies described in this proposal are based on the observation that ARF regulates multiple proliferation-associated proteins. ARF induces proteolysis of E2F1 and DP1; and sequesters c-Myc in the nucleolus. Oncogenic Ha-Ras, which induces cellular senescence in mouse embryonic fibroblasts by stimulating the expression of ARF, causes a relocalization of DP1 in the nucleolus. The objectives of the proposal are to investigate the mechanism by which ARF regulates E2F/DP1 and test the hypothesis that the E2F/DP1-regulatory function is related to the tumor suppression and the senescence-induction functions of ARF. Since the E2F-family of factors (E2Fs) are key mediators of oncogenic activation and cell-proliferation, our studies will establish a novel and potentially dominant mechanism of tumor suppression. Also, the studies will establish a role of E2F/DP1-regulation in cellular senescence induced by ARF. The hypotheses stated above will be investigated through the following aims: 1. Is the DP1- regulatory activity required for the cell cycle-inhibition by ARF? 2. How does ARF induce proteolysis of DP1 and E2FI? 3. What is the role of the E2F/DP1-regulation in the ARF-induced tumor suppression and senescence?